Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit

Abstract

Breast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits.

Figure 1

BCAR4 expression in wild-type rabbit (a) in a panel of somatic organs and in the ovary at adulthood, at 1 month old and 2 days old; (b) in growing follicles; (c) in oocyte and follicle somatic cells (the RPL19 panel is a composite of non-adjacent lanes from a single gel); (d) throughout early embryo development (mean ± SEM). (−) indicates the negative control.

Figure 2

Expression of BCAR4 in preantral follicles of wild-type and genome-edited animals. Relative transcript abundance, mean ± SEM from analysis of 6 +88/+88 does and 5 +88/N and N/N does. Different superscripts indicate significant differences.

Figure 3

Impact of the genotype onto female fertility. (a) Delivery rate and (b) prolificacy. (mean ± SEM). Different superscripts indicate significant differences. Analysis from 7 +88/N does and 6 +88/+88 and N/N does.

Figure 4

Ovarian physiology. (a) AMH concentration in adult female plasma; (b) Ovarian weight (sum of left and right ovaries); (c) Number of corpora lutea after ovarian stimulation; (d) number of haemorrhagic follicles; (e) Density of follicles in the ovarian cortex. Mean±SEM from 5 (except e, 6) +88/+88 does, 6 (except b, 4) +88/N does and 4 (except b, 3 and e, 5) N/N does.

Figure 5

BCAR4 gene edition. BCAR4 +88 allele was generated by two insertions of 2 and 16 nucleotides (in blue), a deletion of 63 nucleotides including the first 19 putative codons (in red), and insertion of a fragment of 133 nucleotides (in green) duplicated from an upstream genomic region. The arrow indicates the intron-exon junction.